Overexpression the BnLACS9 could increase the chlorophyll and oil content in Brassica napus

Background Chlorophyll is a very important pigment involved in photosynthesis, while plant acyl-CoA biosynthesis is derived from plastid-localized fatty acids (FAs). Until now, the regulation of the acyl-CoA pathway for chlorophyll biosynthesis is still unknown. Results Here, we identified a long-chain acyl-CoA synthetase (LACS) gene BnLACS9 from Brassica napus. BnLACS9 complemented a LACS-deficient yeast strain YB525, which indicated that BnLACS9 has the LACS function. BnLACS9 was localized in the chloroplast envelope membrane, while mainly expressed in young leaves and flowers. Overexpression of BnLACS9 in Nicotiana benthamiana resulted in an increase in total CoA and MGDG content. In B. napus with overexpression of BnLACS9, the number of chloroplast grana lamellae and the chlorophyll content, as well as the MGDG and DGDG contents, increased compared to wild type. The net photosynthetic rate, dry weight of the entire plant and oil content of seeds increased significantly, accompanied by an increase in chlorophyll content. Transcriptome analysis revealed that overexpression of BnLACS9 improved the pathway of acyl-CoA biosynthesis and further improved the enzymes in the glycolipid synthesis pathway, while acyl-CoA was the substrate for glycolipid synthesis. The increased glycolipids, especially MGDG and DGDG, accelerated the formation of the chloroplast grana lamellae, which increased the number of chloroplast thylakoid grana lamella and further lead to increased chlorophyll content. Conclusions In the present study, we demonstrated that BnLACS9 played a crucial role in glycolipids and chlorophyll biosynthesis in B. napus. The results also provide a new direction and theoretical basis for the improvement of the agronomic traits of plants. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-022-02254-3.

There are multiple LACS genes in plants, which encode enzymes that perform different roles in lipid metabolism [4,[6][7][8]. In Arabidopsis, nine genes (AtLACS1, AtLACS2, AtLACS3, AtLACS4, AtLACS5, AtLACS6, AtLACS7, AtLACS8, AtLACS9) that encode LACS have been identified [4,9]. Amino acid sequence analysis  16:3 found they all have one very highly conserved motif, the AMP-binding protein (AMPBP) superfamily [10]. Seven of the nine genes could complement the growth phenotype of a LACS-deficient yeast strain, YB525, except AtLACS6, and AtLACS7 [4,11]. AtLACS1 and AtLACS2 were located in ER and the AtLACS1 has overlapping functions with AtLACS2 in wax and cutin synthesis in Arabidopsis thaliana [12][13][14]. Dirk Jessen found that AtLACS1 and AtLACS4 play a synergistic effect in the proper formation of the pollen coat in Arabidopsis [15]. Furthermore, AtLACS4 could catalyze the first step in conversion of peroxisomal indole-3-butyric acid to IAA [16]. AtLACS6 and AtLACS7 play important roles in activating FAs for β-oxidation in the peroxisome [17,18]. It is known that AtLACS9 exists in the chloroplast, a major contributor to chloroplastic LACS activity, involved in the export of plastidial FA export for TAG formation, and its function partially overlaps with LACS1 and LACS4 in Arabidopsis seed oil biosynthesis [19][20][21]. However, the function of AtLACS9 is still controversial, a study showed that AtLACS9 might help transport lipids from the ER back to the plastid [22]. Brassica napus (B. napus) is a worldwide oil crop, which is one of the important edible oils for human consumption and as a raw material for the biofuel and pharmaceutical industry [23][24][25]. Therefore, increasing the oil content of seeds is very important for geneticists and breeders, and has become a major subject of oil crop research [26][27][28]. In B. napus, LACSs play pivotal roles in lipid biosynthesis and oil accumulation [7,[29][30][31]. Pongdontri et al. reported that ACS6 was strongly expressed in embryos of rapeseed and could improve the efficiency of lipid synthesis [30]. The heterologous expression of the BnLACS4 gene in yeast cells could increase the content of C16: 0 and C18: 0 by 45.7 and 21.7%, respectively [31]. Overexpression of BnLACS2 in yeast and rapeseed could increase oil content, and BnLACS2 was located in the ER [29]. Xiao et al. identified 34 BnLACSs by a comprehensive genome-wide analysis of the gene family in B. napus. Comparative expression analysis between highand low-oil B. napus cultivars revealed that BnaLACS6-4, BnaLACS9-3, and BnaLACS9-4 may be involved in chloroplast fatty acid synthesis, and BnaLACS1-10 and 4-1 may play a vital role in lipid biosynthesis [7]. However, except BnLACS2 and ACS6, the functions of other LACS genes are still not very clear in B. napus.
In this study, the BnLACS9 was isolated from developing rape embryos, its cDNA encoding a novel acyl-CoA synthase. Our results showed that overexpression of BnLACS9 in Nicotiana benthamiana can increase the content of galactolipids in the leaf accompanied by an increase in chlorophyll content. Overexpression of BnLACS9 in B. napus also caused the chlorophyll content to be upgraded significantly compared with the wild type. So, our data provide a new insight into the pathway of chlorophyll biosynthesis. The BnLACS9 can regulate the content of the chlorophyll by influencing the chloroplast biosynthesis through regulating the chloroplast functional lipid biogenesis. The photosynthetic rate is directly related to the chlorophyll content of the plant, so we take a new way to increase the biomass of B. napus by enhancing the expression of the BnLACS9 gene in some way.

BnLACS9 is highly homologous with AtLACS9
In Arabidopsis thaliana, nine long-chain acyl-CoA synthetases (LACSs) belong to a large superfamily of acyl-activating enzymes [4,9], and are involved in FA transport. To search the homologous proteins in B. napus L., the amino acid sequences of AtLACS1-9 (AT2G47240, AT1G49430, AT1G64400, AT4G23850, AT4G11030, AT3G05970, AT5G27600, AT2G04350, AT1G77590) were used as the query probe to search in Brassica genome sequences databases (http:// www. genos cope. cns. fr/ blat-server/ cgi-bin/ colza/ webBl at). In total, 29 significant homologous proteins were obtained (Additional file 5: Table S1). The phylogenetic tree analysis was performed based on the similarities of the conserved domain sequences among these proteins. As shown in Fig. 1A, except AtLACS3, other AtLACSs have 2 or 4 homologous proteins in B napus, because the B. napus is allopolyploidy, and it has a duplicate genome [32]. All LACS proteins could be divided into three branches, LACS1 to LACS5 formed the first branch, LACS6 and LACS7 formed the second branch, and LACS8 and LACS9 formed the third branch (Fig. 1A), showing that these LACS proteins could have a different function. The phylogenetic tree analysis showed that AtLACS9, BnaA07g20920D (BnLACS9-A07), and BnaC06g20910D (BnLACS9-C06) were in one clade (Fig. 1A), and they share 91.6% and 91.9% similarity in amino acid sequence, respectively (Fig. 1B). AtLACS9, BnLACS9-A07 and BnLACS9-C06 are closely related in terms of amino acid sequence and evolutionary relationship, which implies that they likely have similar functions. The similarity between BnLACS9-A07 and BnLACS9-C06 was as high as 99.7%, so we selected BnLACS9-C06 as the target gene in this study.
To determine whether BnLACS9 has LACS, a yeast vector pYES2-BnLACS9 was constructed and analyzed by a yeast complementary expression system. Yeast strain YB525 is a strain with affected LACS activity [33], and this defective yeast cannot grow in the medium only using FA as the sole carbon source. A yeast complementarity test was used to determine the growth of yeast cells transferred into different vectors in a drop-out medium  indicated that BnLACS9 could increase the FA content in N. benthamiana leaves. We also detected the chlorophyll content in N. benthamiana leaves of transient expression of BnLACS9 and its control. We found that within 5 days after the BnLACS9 gene was transferred into N. benthamiana leaves, there was no significant difference in chlorophyll content between the two groups. From the sixth day, the chlorophyll content of N. benthamiana leaves transfected with the BnLACS9 gene was significantly higher than that of wild type, and reached the maximum on the eighth day ( Fig. 2A). Monogalactosyl-diaclyglycerol (MGDG) is one of the main components of the chloroplast photosynthetic membrane [34]. We also detected the MGDG content in the leaves of N. benthamiana between the transient expression of BnLACS9 and its control. The content of MGDG in N. benthamiana leaves transfected with the BnLACS9 gene was significantly higher than that of the control (Fig. 2B). All these results indicated that BnLACS9 could improve the content of FA, MGDG and total chlorophyll.

The expression pattern and subcellular localization of BnLACS9
To better understand the function of BnLACS9, we examine the expression pattern of BnLACS9 in various organs, including the root, stem, young leaf, old leaf, flower, and silique of Ningyou 12 (NY12). We performed quantitative real-time PCR (qPCR) analysis to estimate the level of the BnLACS9 transcript. BnLACS9 is mainly expressed in young leaves and flowers (Additional file 2: Fig. S2). We could detect the expression of the BnLACS9 gene in other organs, but the lowest expression in the old leaf. These results show that the BnLACS9 gene is temporally and spatially expressed.
Localization studies were further carried out on BnLACS9 to understand its mechanism of action. To determine the subcellular location of BnLACS9, GFP (green fluorescent protein) was fused to the C terminus of BnLACS9 under the control of a 35S promoter and the fusion gene was transformed into leaves of N. benthamiana. We found that the expression pattern of BnLACS9-GFP overlapped with the chlorophyll autofluorescence in N. benthamiana leaves ( Fig. 3A-C), suggesting that BnLACS9 may be targeted at the chloroplasts. To further study the location of BnLACS9-GFP in chloroplasts, we expressed 35S:BnLACS9-GFP in N. benthamiana leaves for subsequent protoplast isolation and observed the protoplasts of N. benthamiana. From Fig. 3E-G, we observed circular structures of the fusion protein fluorescence signal around the chloroplast, but did not overlap with the spontaneous fluorescence signal of the chloroplast, and even formed loop structures and thin tubules from the chloroplast. With all these observations, we could conclude that BnLACS9 might be located on the chloroplast envelope membrane.

Overexpression of BnLACS9 in B. napus increased the content of the oil content
Previous studies showed that the BnLACS9 gene has an activity of LACS protease (Additional file 1: Fig. S1) and could increase the FA content in N. benthamiana leaves (Additional file 6: Table S2). To study the BnLACS9 gene in rapeseed plant, transgenic rapeseed plants overexpressing BnLACS9 were generated (Additional file 3: Fig. S3). Homozygous BnLACS9-overexpressed lines (#6, #12 and #18) were selected from the positive transgenic plants to evaluate the function of BnLACS9.
The oil content of BnLACS9-overexpressed plant seeds was determined by nuclear magnetic resonance (NMR). As shown in Fig. 4, the oil content of NY12 was only 39.7%, while the oil content of the three overexpression lines exceeded 40%, of which the oil content of BnLACS9-12 reached 45.64%. In addition, the seed oil content of overexpressed BnLACS9 plants was determined by near-infrared (NIR) (Additional file 7: Table S3), and the results also showed the same with NMR. The results of the two methods were consistent, and the oil content of rape seeds overexpressing BnLACS9 increased.

Overexpression of BnLACS9 in B. napus could enhance the content of total chlorophyll
The oil content of BnLACS9-overexpressed plant seeds was increased, we also found that the cotyledons and leaves of BnLACS9-overexpressed plants were significantly greener than those of the wild type (Fig. 5A, B). The chlorophyll content of BnLACS9-overexpressed plants was also higher than that of wild type in 7-day-old cotyledons and 30-dayold leaves (Fig. 5C, D). We also found that the siliques of BnLACS9-overexpressed plants could delay senescence compared with the control (Fig. 5E, F). So, we measured the chlorophyll content of the silique at 30 DAF (days after flowering) and 50 DAF and showed that the chlorophyll content in overexpressed plants was significantly higher than that in wild-type plants (Fig. 5G, H). These results indicated that overexpression of BnLACS9 gene can increase chlorophyll content and delay silique senescence in B. napus.   In order to characterize the mechanism that BnLACS9 influenced the content of chlorophyll through regulating the genes in the pathway of the chlorophyll synthesis, we sequenced the transcriptome of the leaves of the overexpression of BnLACS9 plants and wild type. The unigene was shown as 'comp…_c0' . The expression level of the unigenes was represented by RPKM (Reads Per Kilobase per Million mapped reads) [37]. Transcriptome analysis showed 9 genes (comp613754_c0, comp879850_c0, comp44567_ c0, comp956830_c0, comp55669_c1, comp58218_c0, comp57696_c0, comp38830_c0, comp57073_c0) in chloroplast synthesis pathway were up-regulated in BnLACS9overexpressed plants (Fig. 6). We confirmed the transcript levels of genes HEMA (Glutamyl tRNA reductase), CHLD (Mg-chelatase D subunit), PORB (NADPH-protochlorophyllide oxidoreductase B), CAO (Chlorophyll a oxygenase) by RT-PCR (reverse transcription-polymerase chain reaction, RT-PCR) (Fig. 6B), and showed that these genes were all up-regulated. These results suggested the overexpression of BnLACS9 upgraded the key genes of the chlorophyll synthesis pathway and further increased the content of the chlorophyll.

Overexpression of BnLACS9 in B. napus could increase the number of thylakoid layer structures in chloroplast and the content of the galactolipids
The BnLACS9-overexpressed plants had higher chlorophyll content (Fig. 5), so we observed the chloroplast structure by transmission electron microscopy (TEM). Ultrastructural observation showed that the number of thylakoid grana slice layers in BnLACS9-overexpressed plants was more than that in wild-type plants (Fig. 7). There were fewer layers of thylakoid grana (2-3 layers) were observed in NY12, while more layers of thylakoid grana (5-8 layers) were observed in plants overexpressed with BnLACS9 (Fig. 7). These results indicated that overexpression of BnLACS9 gene could increase the number of thylakoid grana slice layer, make leaves greener and have higher chlorophyll content.
Previous studies showed that the main lipid component of the thylakoid layer structure such as monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQDG) [2,35,36]. Therefore, we used thin-layer chromatography to analyze the lipid content in leaves. The contents of MGDG, DGDG, PG, and SQDG in BnLACS9-overexpressed lines were higher than those in wild type, only PG was not detected in BnLACS9-overexpressed line 6 (Additional file 4: Fig. S4).
In order to make a thorough inquiry about how the content of the glycolipid was increased in the overexpression of BnLACS9 plants and to elucidate the gene regulatory network of the pathway. Transcriptomes in the overexpression of the BnLACS9 line and wild type were analyzed. The unigenes related to glycolipid synthesis were picked from the transcriptome data of B. napus. The unigene was shown as 'comp…_c0' . Transcriptome studies showed that most unigenes of glycolipid biosynthesis were up-regulated in the BnLACS9-overexpressed  (Fig. 8B). In particular, the expression level of the ATS1 and SLS is four times higher more than the CK; and other genes in overexpression of BnLACS9 plant is exceeded double than the CK (Fig. 8B), these results were similar with transcriptomes data. Taken together, the results suggest that BnLACS9 regulated the expression of genes in the pathway of glycolipid biosynthesis. The higher expression level of these genes increased the content of the glycolipid.

Overexpression of BnLACS9 in B. napus could increase photosynthetic efficiency and dry weight of rapeseed
Previous studies have shown that overexpression of BnLACS9 could improve the chlorophyll content in rapeseed (Fig. 5), and increase the expression of genes in the chlorophyll synthesis pathway (Fig. 6). So, we measured the photosynthetic efficiency of leaves of overexpressed plants and wild-type plants. We found that the photosynthetic efficiency of the leaves of the overexpressed BnLACS9 plants was significantly higher than that of wild-type plants (Fig. 9). We measured the dry weight of the BnLACS9-overexpressed lines at 20 and 40 days after seed germination. At 20 days, we could see that the dry weights have been increased. On the 40th day after germination, the dry weight of BnLACS9-overexpressed lines increased significantly (Fig. 10). From the above, overexpression of BnLACS9 in plants could increase photosynthetic efficiency and the biomass of the plant in rapeseed.

Discussion
For a long time, increasing the oil content of oilseed crops has been the core problem of oilseed breeding [25,34,37,38]. LACSs have been show to play a key role in FA and lipid metabolism and could increase oil content [4,11,12,29]. However, there have been few studies on the function of LACS proteins in B. napus, a major oil crop in the world. In this study, we characterized the function of BnLACS9 in B. napus. We showed that BnLACS9 has LACS activity, which could complement a LACS-deficient yeast strain (YB525). Moreover, overexpression of BnLACS9 in B. napus could increase the content of chlorophyll by regulating the pathway of glycolipid synthesis, and then increases lipid content. Therefore, BnLACS9 is a candidate gene for high-oil-content breeding in rapeseed. The B. napus (an allotetraploid) was formed by spontaneous hybridization between B. rape and B. oleracea about 7500 years ago [39], which has a highly homologous genome with Arabidopsis thaliana [40]. Xiao   (BnaLACS9-1, 9-2, 9-3, and 9-4) in rapeseed, however BnaLACS9-1 and 9-2 lost gene function because they obviously different from other BnaLACSs and hardly expressed in all tissues [7]. Our phylogenetic analyses showed that BnaA07g20920D (BnLACS9-A07) and BnaC06g20910D were homologous to Arabidopsis AtLACS9, and share 91.6% and 91.9% similarity in amino acid sequence, respectively (Fig. 1), speculated that they have similar functions. Previous studies have shown that most of the LACS genes have the ability to complement a strain of yeast lacking LACS [4]. Our study showed that heterogeneous expression of BnLACS9 can complement the LACS-deficient yeast mutant YB525, which could rescue with long-chain FAs (C14-C22) as the sole carbon source, but not with short chain FA (C12) (Additional file 1: Fig. S1). These results showed that BnLACS9 has the long-chain acyl-CoA synthetase activity, and also revealed its substrate preference and specificity. It is well known that the function of protein is closely related to its location. In Arabidopsis, previous studies have shown that LACS1, 4 and 8 are located in the ER, LACS6 and 7 are located in the peroxisome, and LACS9 resides in the plastid envelope [4,12,19]. Breuers et al. further research demonstrated the localization of AtLACS9 to the outer envelope membrane [41]. In rice, the OsLACS9 was located in the chloroplast envelope membrane [42]. In this study, subcellular localization analysis indicated that the fluorescence signal of the BnLACS9-GFP fusion protein was around the chloroplast but did not overlap with the spontaneous fluorescence signal of the chloroplast and even formed loop structures and thin tubules from the chloroplast (Fig. 3). The discovery of the loop structure suggests that BnLACS9 has a function at the contact site between organelles, such as the chloroplast and the ER. Therefore, it helps to transport acyl-CoA formed in chloroplasts to ER, and then form TAGs. This is consistent with the fact that ACSL4 is present in the mitochondrial-associated membrane in animal cells [43].
In plant, activation of free FAs to acyl-CoA derivatives is necessary to provide the substrates for glycolipid biogenesis. The synthesis of glycolipid can be divided into chloroplast and endoplasmic reticulum: the inner envelope-localized prokaryotic pathway and the ERlocalized eukaryotic pathway [44]. Chloroplast LACS activity is essential to for glycolipid synthesis, since chloroplasts are the main site of de novo FA synthesis [45]. The AtLACS9, which has been shown to reside in the Fig. 9 The net photosynthetic rate of leaves of BnLACS9 overexpression transgenic lines. *p < 0.05, **p < 0.01. Student's t-test was used to generate the p-value Fig. 10 The dry weight of the seedling from BnLACS9 overexpression transgenic lines. *p < 0.05, **p < 0.01. Student's t-test was used to generate the p-value plastid [46], is considered to be the main LACS isoform involved in the production of acyl-CoA for the biosynthesis of membrane glycerolipids and storage TAGs [47]. The BnLACS9 is belonged to acyl-CoA synthase, transform the FA into acyl-CoA (Fig. 4). Overexpression of BnLACS9 stimulated much more FAs from chloroplast, which are further used for the synthesis of glycerolipids, so that the contents of MGDG, DGDG, PG, and SQDG in BnLACS9-overexpressed lines were higher than those in wild type (Additional file 4: Fig. S4), and BnLACS9overexpressed plants had more thylakoid grana slice layers than wild type (Fig. 7). These results were also confirmed by transcriptome and RT-PCR studies: most glycolipid biosynthesis genes were up-regulated in plants overexpressed with BnLACS9 (Fig. 8). Furthermore, plants overexpressed with BnLACS9 were significantly greener, their chlorophyll content increased (Fig. 5), and chlorophyll synthesis genes were up-regulated (Fig. 6). Therefore, BnLACS9-overexpressed plants have a higher photosynthetic efficiency (Fig. 9), which is conducive to the accumulation of dry matter (Fig. 10) and the formation of oil (Fig. 2). In conclusion, BnLACS9 is a key gene in oil synthesis. In general, BnLACS9 was located in the chloroplast envelope, which could promote the transport of fatty acyl-CoA from the chloroplast to the ER. In turn, the increased fatty acyl-CoA in ER can promote the content of galactolipids in chloroplasts, increase the number of thylakoid grana slice layers and improve the photosynthetic efficiency in the chloroplast, and finally increase the synthesis of TAG (Fig. 11). Therefore, BnLACS9 could increase the oil content of rape seeds.

Plant materials and growth conditions
The seeds of WT rapeseed cultivar 'Ningyou 12 (NY12)' and 'Zhongshuang 11 (ZS11)' as well as N. benthamiana stored in our lab were sown in a soil mix (peat moss/perlite/vermiculite, 5/3/2, v/v/v) in flower pots (4-5 seedlings/pot) and grown in a plant growth room under the following growth conditions: 22 ± 2 °C with a 16 h light: 8 h dark photoperiod at a light intensity of 5000 LX and 60% relative humidity.

RNA extraction, cDNA synthesis, and real-time PCR (RT-PCR)
Total RNA extractions from various tissues and reverse transcription were performed according to Wang et al. with some modifications [50]. For RT-PCR, 25 ng of cDNA was applied using corresponding gene-specific primer pairs. BnACTIN was amplified as the control. RT-PCR was performed on a cycler apparatus (Bio-Rad, USA) using the SYBR Green Master Mix (Vazyme, China) according to the manufacturer's instructions. Amplification was conducted in 96-well optical reaction plates with the following protocol: 94 ℃ for 4 min, 40 cycles of 94 ℃ for 15 s, 58 ℃ for 15 s, and 72 ℃ for 15 s. The relative expression levels were estimated using the 2 −∆∆CT method of Livak and Schmittgen [51]. The RT-PCR was repeated with three biological replications. Primers used in the study are listed in Additional file 8: Table S4.
For transcriptome analysis, 100 mg of the leaf was used for RNA extraction. The purified RNA was detected and qualified using a OneDrop OD-1000 + spectrophotometer (RockGene, Shanghai, China). Library construction was performed according to the standard protocol. The sequencing of the library was performed using the BGISeq-500 platform. The experiment was repeated three times with independent samples. The raw data were filtered to trim adaptor sequences and to remove Fig. 11 The model of the function of BnLACS9. BnLACS9 was located in the chloroplast envelope, which could promote the transport of fatty acid acetyl-CoA from the chloroplast to the ER. In turn, the increased fatty acyl-CoA in ER can promote the content of galactolipids in chloroplasts, increase the number of thylakoid layer structures and improve the photosynthetic efficiency in the chloroplast, and finally increase the synthesis of TAG low-quality sequences (Q < 20) with > 10% uncertain (N) bases. The NCBI database was used to annotate gene function. Differentially expressed genes (DEGs) were screened according to the NOIseq method [52]. Transcripts that reach the criterion of log 2 -fold change ≥ 1 (or ≤ -1) and probability ≥ 0.8 were selected as DEGs.

Subcellular localization of BnLACS9 by transient expression in N. benthamiana leaves and B. napus cotyledons
To determine the expression location of the BnLACS9 protein, the full-length BnLACS9 cDNA was PCR amplified using the primers (BnLACS9-LF/BnLACS9-LR) with the cDNA as a template. The PCR product was cloned into the pENTR ™ TOPO ® vector (Invitrogen, USA), and the clone with correct sequencing was recombined into pK7WG2.0 by Gateway cloning (Invitrogen, USA) to produce GFP fusion proteins. The recombinant plasmid was transformed into the Agrobacterium tumefaciens strain GV3101. Overnight cultured A. tumefaciens strain GV3101 harboring the recombinant vectors were washed with sterile 10 mM MMA (10 mM MES, 10 mM MgCl2, and 100 mM acetosyringone, pH 5.6) and suspended with injection buffer MMA to the OD600 = 1.6. The GV3101 containing the construct 35S::p19 was also cultured and suspended with MMA to the OD600 = 1.6, which expression the Tomato bushy stunt virus p19 protein to suppress the gene silencing [53]. The two cultures were mixed 1:1 to the OD600 = 0.8, and followed by incubation at 28 °C with shaking for 3 h. Agrobacterium infiltration into 3-to 4-week-old N. benthamiana leaves was performed as described previously [54,55]. Plant culture conditions are the same as the N. benthamiana culture conditions as follow: illumination weekly 12 h (light intensity 5000 LX, temperature 24 °C, humidity 60%) and dark 12 h (temperature 22 °C, humidity 60%). An important step is that the injected B. napus cotyledons must be maintained humidity in a film bag. The protoplasts from N. benthamiana leaves were performed as described previously [41]. Four to seven leaf disks with a diameter of 0.8 cm of transfected were cut with a cork borer and transferred into a 10-ml syringe containing 2 ml of cell wall digestion solution (CWDS: 1.5% cellulase R-10, 0.4% macerozyme R-10, 0.4 M mannitol, 20 mM KCl, 20 mM MES (pH 5.6), 10 mM CaCl2, 0.1% BSA). Then, protoplasts and plant tissue were monitored by confocal microscopy (Leica TCS SP5, Wetzlar, Germany). The fluorescence emissions were at 510-540 nm for eGFP and 658-665 nm for chloroplast.

Plant expression vector constructs and A. tumefaciens-mediated plant transformations
To construct the expression vector containing the BnLACS9, the full-length BnLACS9 cDNA was PCR amplified using the primers (BnLACS9-LF and BnLACS9-NR) with the cDNA as a template. The PCR product was cloned into the pENTR ™ TOPO ® vector (Invitrogen, USA), and the clone with correct sequencing was recombined into the vector pB2WG7.0 by the gateway (Invitrogen, USA) to create the vector under the control of the constitutive Cauliflower mosaic virus (CaMV) 35S promoter. The expression vector contained in the BnLACS9 was transformed into the A. tumefaciens strain GV3101. For transient expression in N. benthamiana leaves, Agrobacterium infiltration into 3-to 4-weekold N. benthamiana leaves was performed as described previously [54,55].
To construct a vector for the constitutive expression of BnLACS9, a full-length BnLACS9 (BnLACS9-OF/BnLACS9-OR) cDNA was PCR amplified from its cDNA clone, and the CaMV 35S promoter (CaMV 35S-F/CaMV 35S-R) and CaMV Nos terminator (CaMV Nos-F/CaMV Nos-R) was generated from the vector pEGAD. Then, all three products were inserted into the EcoR I/Hind III sites of pCAMBIA1300, creating the pCAMBIA1300-35S-BnLACS9-NOS vector, which was then transformed into GV3101. To construct the vector to suppress BnLACS9 expression, a highly conserved 164-bp cDNA fragment of the 5′-open reading frame was amplified with primers Bnlacs9-F and Bnlacs9-R. This cDNA fragment was subcloned into the pENTR/ D-TOPO vector, creating pENTR::Bnlacs9. Then, the pENTR::Bnlacs9 plasmid was transferred into the destination vector pHellsgate 12 to generate pHellsgate 12::Bnlacs9 using the Gateway LR recombinase (Invitrogen, USA). The plasmid pHellsgate 12::Bnlacs9 was transformed into GV3101. And the two vectors were transformed using an Agrobacterium-mediated transformation method as described by Wang et al. [56]. At least three independent transformants were examined as described in the results section. Hygromycin-resistant T2 generation plants were identified by PCR from the progeny of the primary transformants.

Ultrastructural observation of chloroplasts
The leaves of wild type and BnLACS9-overexpressed lines were cut into 2-mm segments and fixed in 2.5% glutaraldehyde solution. Then they were put into the refrigerator at 4 °C for 24 h. The sample was replaced every 3 days during storage. Fixed samples were sent to the Electron Microscope Center of Nanjing Academy of Agricultural Sciences for sample preparation, and the ultrastructure of chloroplasts was observed by transmission electron microscopy (TEM). TEM sample preparation methods: samples were fixed in 3% glutaraldehyde and 0.1 mol/L phosphate buffer (pH 7.2) for 4 h after collection, then fixed in 1% osmium acid (pH 7.2) for 4 h after washing, and then embedded and polymerized with SPURR after dehydration of acetone step by step. After the dehydration of ethanol step by step, the embedded samples were sliced and then dried at the critical point with by isoamyl acetate. The samples were observed and photographed by JEM-1230 transmission electron microscopy (JEOL LTD, Japan).

Chlorophyll determination
Leaves were crushed into powder in liquid nitrogen and then homogenized in 80% acetone, and the debris was removed by centrifugation at 12,000 rpm for 5 min. The absorbance of the supernatant at 663 and 645 nm was measured using a spectrophotometer (GE Healthcare, USA). The chlorophyll A and chlorophyll B concentration of the samples was determined as described previously [59].

Photosynthetic activity
Photosynthetic parameters of the plants were measured using a portable photosynthesis system (LI-6400XT, LI-COR, http:// www. licor. com/) in the field. Measurements were performed in the morning (9:00, 11:00 AM and 15:00 PM). All data represent the means obtained from five plants in one line.
Lipid detection was carried out by spraying the plate with 5% sulfuric acid in the water, followed by charring at 180 °C for 5 min, or exposing the TLC plate to iodine vapor, for staining all classes of lipids. The estimation of the content of individual polar and neutral lipids of the total lipid extracts was performed by video densitometry analysis of spots on TLC, obtained after averaging three replicates of C, NI, and NII (ImageJ software).